The following must be addressed in the protocol:-

                1: Important -> large number of regeneratable cells. This is usually

                    maximised by including a tissue culture step.

                2: Optimising plant and Agrobacterium genotype-

                    It is not essential to transform "elite" lines but close relative that may be

                    crossed in. e.g. diploid wild strawberry -> Fragaria vesca ->cross with octoploid

                    edible strawberry.

                3: Adding vir gene inducers such as acetosyringone

                4: pH, temperature, light conditions

                5: Length of co-cultivation

                6: Hormones

                7: Choice of explant type

                8: Is wounding necessary? Ultra-sound may improve transformation

        Transforming tobacco leaf disks (see slide show)

        Transformation in planta: Arabidopsis using floral dip.

        First tried because egg cell receives entire genome from sperm cell – Is it therefore also receptive to
         T-DNA insertion?

        Possible targets: gametophyte or recently fertilised embryos.

        Chop back first flowering: Dunk when second flowering "bolt" is 2->10 cm high.

        Needs no hormones or culture steps; no problem with somaclonal variation

        Needs a "surfactant" e.g. Silvet 77:

        Enhances entry of bacteria into inaccessible plant tissues-

        New techniques are being developed where Arabidopsis plants are simply sprayed with
        Agrobacterium.

        Few plant cells are terminally differentiated and are totipotent:
 
         Two forms of plant tissue culture:

         ORGANOGENESIS AND SOMATIC EMBRYOGENESIS (more on the latter later).
 

       Organogenesis

         Relies on the ability ot most cells to "dedifferenetiate" and revert to a being "stem-cell".

         i.e. actively dividing with no clear viariation in cell type.

         In plants, this is refered to as formation of MERISTEMS and in culture is typhified by the

        formation of callus tissue.

         Various explants or suspension cell cultures can be used to induce meristemic tissue when cultures

          with hormones (see handout).

         By changing the in hormones in the culture medium it is possible to induce callus cultures to

        re-differentiate to form viable plants.

         A Problem: Somaclonal variation

        Tissue culture involves successive rounds of plant division.

        With increasing time in culture, the regenerated plant displays abnormalities  e.g. albinism.

•         Genetic analysis revealed considerable chromosomal re-arrangements.
•         Not due to "active division" as not observed with meristematic tissue.
•         But callus tissue is also stressed.
•         Non-physiological levels of hormones are used.

 
       But What is the cause of this somaclonal variation?

       Two main mechanisms are suspected..

•         Plant genomes are full of repetitive DNA.

•         This repetitive DNA contains many examples of mobile elements. The function of many

•        Recombination between homologous regions.
•        Transposons and retro-elements becoming activated and mutating genes?

      Surprisingly, certain plant tissues can be induced to form "somatic embryos" which are fundamentally

     very similar to "zygotic embryos" which form within the seed. 

      A very attractive feature as

•         Makes regeneration easy - simply involving the "germination" of the embryos.

•         No problem with somaclonal variation.

    Zygotic Embryo Development

    Covers development from the time of fertilisation until seed formation occurs.

    The major roles for the embryogenic process are:

    1. Establish the basic body plan.

    2. Set aside meristematic tissue for postembryonic elaboration of structure (leaves, roots,

        flowers, etc.).

    3. Establish an accessible food reserve for the germinating embryo until it becomes autotrophic.

 

    The basic body plan begins with (see above diagram)

1. A asymmetrical cell division giving rise to the terminal cell and the basal cell.
2. The terminal cell gives rise to the embryo proper.
3. The basal cell forms closest to the micropyle.
4. It gives rise to the suspensor.

•     Orientates the absorptive surface of the embryo toward its food source
•     Serves as a nutrient conduit for the developing embryo.

1. The distal end of the suspensor is called the hypophysis and in many species it gives rise to the root meristem.
2. Axial and radial patterns develop to form a globular-stage embryo with tiers of cells. The emerging shape of the embryo depends on regulation of the patterns of cell division.
3. Radial patterning emerges in the globular stage as the three tissue systems (dermal, vascular, and ground) of the plant are initiated.

•     The dermal system will form from the protoderm and contribute to the outer protective layers

•       The vascular system functions in support and transport and arises from the procambium cells that

•     Ground tissue forms from the ground meristem and surrounds the developing vascular tissue;

• Cotyledons may aid in nourishing the plant by becoming photosynthetic after germination.
• The shoot apical meristem and root apical meristem are clusters of embryonic cells that persist in postembryonic development and give rise to most of the actual plant.
• The shoot apical meristem will initiate leaves and ultimately the transition to reproductive development after germination. When does this form? In Arabidopsis the cotyledons are produced from general embryonic tissue, not from the shoot meristem
• Position rather than clonal origin appears to be the critical factor in embryo pattern formation, implicating some type of cell-cell communication, e.g. microsurgery experiments on somatic carrot embryos demonstrate that isolated pieces of embryo can often replace the missing complement of parts.

            

        Immature embryos are a very good source of embryogenic tissue.

        Embyros also form near to the surface; easy to access for transformation (see below). 

        Auxins: especially good at inducing embryo formation.

        Culture on auxin free medium to initiate root formation. .

        Certain plants are very difficult to transform using Agrobacterium. An alternative DNA involves

       "bombardment " of  somatic embryogenic callus with naked DNA containing the transgene of

        interest  and a selectable marker. This is a very inefficent process depending on the transgene

        penetration of the plant nucleus and insertion into the plant genome.  Nevertheless, this is the

        current technique of choice when transforming cereal plants.
 

        The DNA coated onto tungsten / gold beads..Microcarriers 

        The DNA/microcarroers are accelerated on to plant/ explant/ callus samples using gunpowder,

        carbon dioxide, electric charge or helium.

        Using 12 separate plasmids up to 600kb DNA can be introduced at once particle bombardment.

           Optimised by

•         Pre-culture of explant material..By far the most important step.
•         Small size of microcarriers (0.2-0.4mm)
•         Osmotic pre-treatment

•          Very inefficient as relies on DNA inserting into the nucleus and being integrated into "host"
•            genome.
•          Complex DNA integration pattern.
1.   Ligation / rearrangement of DNA fragments before integration.
2.   Co-suppression may result.

        1. Agrolistics : A half way house between Agrobacterium and biolistics

        2. Protoplast fusion (see slide-show to see what protoplasts look like)

        -         Cell wall is a significant barrier to “naked” DNA uptake.
        -         Most “fussy” technique.
        -         Mechanical and/or enzymatic digestion
        -         Cellulase
        -         Pectic lyase
        -         Cocktail enzymes “Macerozyme”
        -         Some cell –types are more competent for DNA uptake than other.
                  e.g. sugar beet; enrichment for guard cell protoplasts by differential centrifugation.
        -        DNA uptake: Involves perturbation of the plasma membrane

                        1. Polyethylene glycol.- amphipathic molecule which alters the mobility of the
                            membrane phospholipids.

                        2. Electroporation – Use electric charge to “punch” hole in membrane

                        3. Liposomes.